Delving into defence: identifying the Pseudomonas protegens Pf-5 gene suite involved in defence against secreted products of fungal, oomycete and bacterial rhizosphere competitors

Competitive behaviours of plant growth promoting rhizobacteria (PGPR) are integral to their ability to colonize and persist on plant roots and outcompete phytopathogenic fungi, oomycetes and bacteria. PGPR engage in a range of antagonistic behaviours that have been studied in detail, such as the production and secretion of compounds inhibitory to other microbes. In contrast, their defensive activities that enable them to tolerate exposure to inhibitory compounds produced by their neighbours are less well understood. In this study, the genes involved in the Pseudomonas protegens Pf-5 response to metabolites from eight diverse rhizosphere competitor organisms, Fusarium oxysporum, Rhizoctonia solani, Gaeumannomyces graminis var. tritici, Pythium spinosum, Bacillus subtilis QST713, Pseudomonas sp. Q2-87, Streptomyces griseus and Streptomyces bikiniensis subspecies bikiniensi, were examined. Proximity induced excreted metabolite responses were confirmed for Pf-5 with all partner organisms through HPLC before culturing a dense Pf-5 transposon mutant library adjacent to each of these microbes. This was followed by transposon-directed insertion site sequencing (TraDIS), which identified genes that influence Pf-5 fitness during these competitive interactions. A set of 148 genes was identified that were associated with increased fitness during competition, including cell surface modification, electron transport, nucleotide metabolism, as well as regulatory genes. In addition, 51 genes were identified for which loss of function resulted in fitness gains during competition. These included genes involved in flagella biosynthesis and cell division. Considerable overlap was observed in the set of genes observed to provide a fitness benefit during competition with all eight test organisms, indicating commonalities in the competitive response to phylogenetically diverse micro-organisms and providing new insight into competitive processes likely to take place in the rhizosphere.

Characterization and aggressiveness of take-all root rot pathogens isolated from symptomatic bermudagrass putting greens

Take-all root rot is a disease of ultradwarf bermudagrass putting greens caused by Gaeumannomyces graminis (Gg), Gaeumannomyces sp. (Gx), Gaeumannomyces graminicola (Ggram), Candidacolonium cynodontis (Cc), and Magnaporthiopsis cynodontis (Mc). Many etiological and epidemiological components of this disease remain unknown. Improving pathogen identification and our understanding of the aggressiveness of these pathogens along with growth at different temperatures will advance our knowledge of disease development to optimize management strategies. Take-all root rot pathogens were isolated from symptomatic bermudagrass root and stolon pieces from 16 different golf courses. Isolates of Gg, Gx, Ggram, Cc, and Mc were used to inoculate ‘Champion’ bermudagrass in an in planta aggressiveness assay. Each pathogen was also evaluated at 10, 15, 20, 25, 30, and 35C to determine growth temperature optima. Infected plant tissue was used to develop a real-time PCR high resolution melt assay for pathogen detection. This assay was able to differentiate each pathogen directly from infected plant tissue using a single primer pair. In general, Ggram, Gg, and Gx were the most aggressive while Cc and Mc exhibited moderate aggressiveness. Pathogens were more aggressive when incubated at 30C compared to 20C. While they grew optimally between 24.4 and 27.8C, pathogens exhibited limited growth at 35C and no growth at 10C. These data provide important information on this disease and its causal agents that may improve take-all root rot management.

Efficient Biocontrol of Gaeumannomyces graminis var. Tritici in Wheat: Using Bacteria Isolated from Suppressive Soils

“Take-all” disease is the most important biotic factor affecting cereal productivity, causing 30–50% of crop losses. The causal agent is the ascomycete soil-borne pathogen Gaeumannomyces graminis var. tritici (Ggt). Current control measures are ineffective, because Ggt can remain saprophytic in soils for long periods. Therefore, the study of the microbiome residing in suppressive soils (SS) is a promising niche of Ggt biocontrol. Here, we evaluated the efficiency of Serratia sp., Bacillus sp., and Acinetobacter sp. isolated from SS against the incidence of Ggt on wheat. Our results demonstrated that plants inoculated with the bacterial consortium in both greenhouse and field conditions were highly efficient in Ggt biocontrol, more so than individual strains. The disease reduction was evidenced by higher biomass production, fewer copies of the Ggt genome with a concomitant curtailment of blackening of roots, a decrease of lipid peroxidation, and an increase of superoxide dismutase activity. The ability of the microbial consortium over that of single strains could be attributable to interspecies communication as a strategy to biocontrol; i.e., higher chitinase activity. In conclusion, bacterial consortia from SS are an important niche of Ggt biocontrol, serving as a model for other soil-borne pathogens.

4-Ethylphenol, A Volatile Organic Compound Produced by Disease-Resistant Soybean, Is a Potential Botanical Agrochemical Against Oomycetes

Oomycetes, represented by Phytophthora, are seriously harmful to agricultural production, resulting in a decline in grain quality and agricultural products and causing great economic losses. Integrated management of oomycete diseases is becoming more challenging, and plant derivatives represent effective alternatives to synthetic chemicals as novel crop protection solutions. Biologically active secondary metabolites are rapidly synthesized and released by plants in response to biotic stress caused by herbivores or insects, as well as pathogens. In this study, we identified groups of volatile organic compounds (VOCs) from soybean plants inoculated with Phytophthora sojae, the causal agent of soybean root rot. 4-Ethylphenol was present among the identified VOCs and was induced in the incompatible interaction between the plants and the pathogen. 4-Ethylphenol inhibited the growth of P. sojae and Phytophthora nicotianae and had toxicity to sporangia formation and zoospore germination by destroying the pathogen cell membrane; it had a good control effect on soybean root rot and tobacco black shank in the safe concentration range. Furthermore, 4-Ethylphenol had a potent antifungal activity against three soil-borne phytopathogenic fungi, Rhizoctonia solani, Fusarium graminearum, and Gaeumannomyces graminis var tritici, and four forma specialis of Fusarium oxysporum, which suggest a potential to be an eco-friendly biological control agent.

Biological Control of Take-All and Growth Promotion in Wheat by Pseudomonaschlororaphis YB-10

Wheat is a worldwide staple food crop, and take-all caused by Gaeumannomyces graminis var. tritici can lead to a tremendous decrease in wheat yield and quality. In this study, strain YB-10 was isolated from wheat rhizospheric soil and identified as Pseudomonas chlororaphis by morphology and 16S rRNA gene sequencing. Pseudomonas chlororaphis YB-10 had extracellular protease and cellulase activities and strongly inhibited the mycelium growth of Gaeumannomyces graminis var. tritici in dual cultures. Up to 87% efficacy of Pseudomonas chlororaphis YB-10 in controlling the take-all of seedlings was observed in pot experiments when wheat seed was coated with the bacterium. Pseudomonas chlororaphis YB-10 was also positive for indole acetic acid (IAA) and siderophore production, and coating wheat seed with the bacterium significantly promoted the growth of seedlings at 107 and 108 CFU/mL. Furthermore, treatment with Pseudomonas chlororaphis YB-10 increased activities of the wheat defense-related enzymes POD, SOD, CAT, PAL and PPO in seedlings, indicating induced resistance against pathogens. Overall, Pseudomonas chlororaphis YB-10 is a promising new seed-coating agent to both promote wheat growth and suppress take-all.

Activity of Fengycin and Iturin A Isolated From Bacillus subtilis Z-14 on Gaeumannomyces graminis Var. tritici and Soil Microbial Diversity

Bacillus subtilis Z-14 can inhibit phytopathogenic fungi, and is used as a biocontrol agent for wheat take-all disease. The present study used the soil-borne fungus Gaeumannomyces graminis var. tritici (Ggt), which causes wheat take-all disease, and the soil microbial community as indicators, and investigated the antifungal effects of fengycin and iturin A purified from strain Z-14 using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, respectively. The results showed that fengycin destroyed the internal structure of Ggt cells by digesting the cytoplasm and organelles, forming vacuoles, and inducing hyphal shrinkage and distortion. Iturin A induced cell wall disappearance, membrane degeneration, intracellular material shrinkage, and hyphal fragmentation. A biocontrol test demonstrated a 100% control effect on wheat take-all when wheat seedlings were treated with fengycin at 100 μg/ml or iturin A at 500 μg/ml. Iturin A and fengycin both reduced the relative abundance of Aspergillus and Gibberella. At the genus level, iturin A reduced the relative abundance of Mortierella and Myrothecium, while fengycin reduced that of Fusarium. Only fengycin treatment for 7 days had a significant effect on soil bacterial diversity.

First Report of Fusarium culmorum and Microdochium bolleyi Causing Root Rot on Triticale in Kazakhstan

Triticale (×Triticosecale Wittmack) is obtained from wheat × rye crossing. It is positioned between wheat and rye in terms of resistance to soilborne pathogens including Gaeumannomyces graminis var. tritici, Fusarium culmorum, F. avenaceum, and Bipolaris sorokiniana (Arseniuk and Góral 2015). In 2019, seven triticale fields were surveyed in Almaty Province, Kazakhstan to examine soil-borne fungal pathogens. A total of 28 symptomatic plants with stunting, rot or discolored root were collected to identify causal agents. The overall disease incidence was approximately 8 to 10% in the fields. Fungi were isolated from 3-5 mm pieces excised from symptomatic tissues. The pieces were exposed to surface disinfection in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, blotted dry, and plated on 1/5 strength potato dextrose agar (PDA) amended with 0.01% streptomycin. Plates were left in the dark at 23°C for 7 days. A total of 34 fungal colonies were isolated of which nineteen isolates, originally from six fields showed the cultural characteristics of B. sorokiniana. This species was previously reported to cause common root rot on triticale in Kazakhstan (Özer et al. 2020). Ten isolates from four fields produced pale orange and cottony mycelium with red pigmentation on the agar, which is typical of Fusarium-like growth. The remaining isolates (n=5) from two fields produced salmon-colored and scarce aerial mycelium with no soluble pigmentation, similar to Microdochium spp. Fusarium isolates produced thick-walled and curved macroconidia with 3-4 septa (n=50, 25.7 to 37.6 × 4.1 to 7.3 μm in size) and notched basal cell on PDA, but microconidia were absent, which matches the description of F. culmorum (Wm.G. Sm.) Sacc. (Leslie and Summerell 2006). Microdochium isolates produced swollen, brown, and thick-walled chlamydospores and hyaline, one-celled, and thin-walled conidia (n=50, 5.4 to 9.3 × 1.5 to 3.0 μm in size) formed on ampullate and cylindrical conidiogenous cells on oatmeal agar (OA). These morphological features are consistent with previous observations for Microdochium bolleyi (R. Sprague) de Hoog & Herm.-Nijh. (Hong et al. 2008). To confirm morphological preliminary identifications, the portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified with EF1/EF2 primers (O’Donnell et al. 1998) for representative Fusarium isolates (n=4) for each field. Additionally, the internal transcribed spacer (ITS) of ribosomal DNA was amplified with ITS1/ITS4 primers (White et al. 1990) for representative Microdochium isolates (n=2) for each field. BLASTn queries against NCBI GenBank revealed that the EF1-α sequences of Fusarium isolates (MW311081-MW311084) shared 100% identity with F. culmorum strain CBS 110262 (KT008433). The ITS sequences of M. bolleyi isolates (MW301448-MW301449) matched that of M. bolleyi strain CBS 137.64 (AM502264) with 100% sequence similarity. Pathogenicity test was conducted on pregerminated seeds of triticale cv. Balausa. A plastic pot (17 cm height, 9 cm in diam) was filled with a sterile mixture of vermiculite, peat, and soil (1:1:1, v/v/v). Mycelial plugs (1 cm in diam) were cut from the margin of a growing culture of representative isolates (Kaz_Fus123 and Kaz_Mb01) and placed onto the mixture in the pot. A sterile agar plug was employed as a control treatment. One pregerminated seed was put on the plug and covered with the mixture. The pots were transferred to a growth chamber set at 23 ± 2°C and a photoperiod of 14 hours. The experiment was performed twice using 5 replication pots per isolate. Four weeks after inoculation, discoloration of the crown was observed on all the inoculated roots, whereas no symptoms were observed on the control plants. Koch’s postulates were fulfilled by reisolating and identifying the pathogen based on the morphology described above. This is the first report of M. bolleyi and F. culmorum causing root rot on triticale in Kazakhstan. Although B. sorokiniana is the most primary pathogen that may limit yield in the production of triticale in Kazakhstan, F. culmorum and M. bolleyi have been found to be less frequent and less aggressive pathogens, respectively. Further studies are needed to better understand the potential distribution and impact of these pathogens on triticale.